Fourier Transform Raman spectroscopy (FT-Raman) has been applied for the non-destructive in-situ analysis of pigments on differently colored flower petals of pansy cultivars (Viola x wittrockiana). The main target of the present study was to investigate how far the Raman mapping technique through FT-Raman spectroscopy and cluster analysis of the Raman spectra is a potential method for the direct, in-situ discrimination of flavonoids (flavonols against anthocyanins) and of carotenoids occurring in flowers, using intact and differently colored flower petal of Viola x wittrockiana for this case study. In order to get more information about the reliability of the direct in-situ flavonoid detection by the Raman method, pigments extracts of the petals were separated by thin-layer chromatography (TLC) and investigated by Raman spectroscopy. Hierarchical cluster analysis (HCA) of the Raman spectra from reference pigments (carotenoids, anthocyanins and flavonols), from areas of the flower petals, and from the TLC extracts allowed discriminating the various pigments, in particular flavonoids (flavonols against anthocyanins) and carotenoids. With a two-dimensional Raman mapping technique, which provides a chemical image of the sample under investigation, we determined by cluster analysis the distribution of carotenoids, anthocyanins and flavonols from the outer layer of the petals, and by integrating through suitable spectral regions selected as characteristic markers for particular pigments their relative concentration could approximately be determined. We found a satisfactory correlation between the patterns seen on the visible images and the patterns on the chemical images obtained by Raman mapping. Copyright © 2011 John Wiley & Sons, Ltd.